Project Summary Hematopoietic stem cells (HSCs), which reside in the bone marrow of adult mammals, are capable of producing all erythroid, myeloid and lymphoid blood cell types of the organism throughout life. Coupled with their own capacity for self-renewal, successful transplantation of healthy HSCs is the only therapy currently available that can completely replace and restore the blood system in patients with leukemia and lymphoma. Despite this need, HSCs currently cannot be efficiently created or cultured in vitro, suggesting that extrinsic factors that support their regulation and growth in vivo are lacking from existing protocols. Using the zebrafish model, our group has previously demonstrated that hemodynamic force from blood flow is an essential requirement for HSC production in vertebrate embryos. Based on our substantial preliminary data, I hypothesize that the Yes-associated protein (YAP) transcription factor directly transduces mechanical input from blood flow into gene expression changes in HSCs. This proposal intends to resolve the molecular mechanisms underlying mechanically-activated, YAP- driven HSC production. I will employ chemical, physical and genetic perturbation of shear stress and cyclic stretch in live zebrafish embryos to assess the impact of these individual components of hemodynamic force on HSC production from hemogenic endothelium. This approach will be validated and translated to an in vitro microfluidic system based on iPSC-derived hemogenic endothelium, to demonstrate conservation in mammals in a therapeutically relevant setting. I will also use the zebrafish system to investigate Piezo and Integrin proteins as likely candidates for membrane-localized proteins that link forces from blood flow with YAP activation and transcriptional commitment to HSC development and maintenance. Understanding the molecular mechanisms by which physical forces during embryonic development govern HSC production may improve current efforts to generate or expand HSCs in vitro for therapeutic use.